CN106771119A - IgG antibody chemiluminescence immune detection reagent kits of a kind of anti-Scl 70 and preparation method thereof - Google Patents
IgG antibody chemiluminescence immune detection reagent kits of a kind of anti-Scl 70 and preparation method thereof Download PDFInfo
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- CN106771119A CN106771119A CN201611018773.8A CN201611018773A CN106771119A CN 106771119 A CN106771119 A CN 106771119A CN 201611018773 A CN201611018773 A CN 201611018773A CN 106771119 A CN106771119 A CN 106771119A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a kind of IgG antibody chemiluminescence immune detection reagent kits of anti-Scl 70, the kit includes：The coated magnetic particles of IgG antibody monoclonal antibody of anti-Scl 70, the acridinium ester of mouse anti-human igg mark, Sample dilution, test dilution, the IgG antibodies of anti-Scl 70 calibration product, preexciting liquid, exciting liquid.In addition the invention also discloses a kind of preparation method of the IgG antibody chemiluminescence immune detection reagent kits of anti-Scl 70.Kit of the present invention is easy to operate compared with available reagent box, and sensitivity is high, the advantages of detection range is wide.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of inspection of chemiluminescence immunoassay
Survey Anti-Scl-70 IgG kits and preparation method thereof.
Background technology
Anti-Scl-70 target antigen is nonhistones for alkalescence, is the degradation product of DNA topoisomerase Is, is cell internal reference
With the enzyme of important biomolecule function.Because initially only in systemic scleroderma（SSc）The antibody, and its antigen molecule are found in patient
It is 70KD to measure, therefore referred to as Anti-Scl-70.DNA Topoisomerase -1, it is dense in kernel in caryoplasm.It
Process of loosening with DNA supercoils is relevant, is worked in the duplication and transcription of DNA double chain.This enzyme cuts off DNA, and attached
On the free-end for producing, therefore, whole DNA molecular uncoiling is not required to, and occur only at the region that will be replicated.Once it is a certain
Region duplication or transcription are completed, and DNA double chain is just recombined, topology enzyme r e lease.It is intracellular with important biomolecule function
Key protein.Scl-70 antibody is mainly IgG, and IgA, IgM are more rare.
Systemic scleroderma is a kind of general CTD, with skin and each organ systems (such as intestines and stomach, lung, the heart,
Kidney etc.) the disease damage of inflammation, blood vessel and collagenous fibrosis be main performance, be it is a kind of with vascular lesion and severe fibrosis be spy
The CTD levied.
Anti-Scl-70 is considered as the serum marker antibody of systemic sclerosis, it is positive often with diffusivity cutaneous lesions,
Near-end skin involves, Cardiac Involvemant, accompanying tumors and pulmonary interstitial fibrosis are closely related, it is considered to be the index of prognosis mala.
Detection Anti-Scl-70 can improve the diagnosis of SSc, be conducive to the antidiastole of clinic and improve therapeutic effect.
The main method of clinical detection Anti-Scl-70 IgG be enzyme linked immunosorbent assay, but the method exist it is following
Weak point：
（1）Using 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 as antigen coat apparatus and instead
Container is answered, 12 batches, 6 batches, 8 batches or whole plate first use can only be divided into when in use, it is impossible to carry out independent, single part
Detection；
（2）Quantitative determination reagent type used is more, and each detection reagent will be contained with reagent bottle, and often be made
It is required for changing imbibition nozzle during with a kind of reagent being filled into the micropore of microwell plate respectively, not only reagent bottle species is more, filling
The operation of reagent is also extremely cumbersome；
（3）Lack the corresponding mark to detection information, can only just will appreciate that or know by checking the mark of kit external packing box
The product batch number and term of validity information of detection reagent are known, and the information known is uncontrolled in detection process, with very big
Randomness；
（4）Detection reagent, in open space, easily causes the cross pollution between various reagents and shadow in detection process
Ring the accuracy of testing result；
（5）Using manual operations more than detection process, the dosage of reagent or sample is not bery accurate, and operating process is extremely cumbersome and multiple
It is miscellaneous, bust is susceptible to, the degree of accuracy of testing result and precision are poor；
（6）In the quantity configuration of detection project reagent set and using item number × 48/96 person-portion is above, if necessary to examine
10 projects are surveyed, then the configuration of reagent and the use of number must be 10 × 48/96 person-portions, if only a sample needs detection 10
Different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
The content of the invention
Current Anti-Scl-70 IgG detection techniques have the following disadvantages：Testing cost is high, detection sensitivity is low, detection
The range of linearity is narrow, reappearance is low, can not quantify, complex operation etc..
The present invention discloses that a kind of testing cost is low, sensitivity is high, detection is linear precisely in order to overcome the above shortcoming
Scope is wide, reappearance is high, can quantify, Anti-Scl-70 IgG kits simple to operate and preparation method thereof.It is of the invention first
Chemical luminescence immune analysis reagent box is first prepared, is mainly included：The coated magnetic particle of Anti-Scl-70 IgG monoclonal antibody,
The acridinium ester and Anti-Scl-70 IgG calibration product of mouse anti-human igg mark；Then automatic chemiluminescence immunoassay is utilized
Instrument detects that drafting standard curve is built in computer software, tests actual sample, according to sample luminous value meter to calibration product
Calculate concentration of specimens；Finally confrontation Scl-70 IgG antibody automatic chemiluminescence immunoassay systems carry out performance（Sensitivity, line
Property, precision, interference）Evaluation.
It is of the invention compared with current technology, with advantages below：
1st, present invention selection acridinium ester is used as marker material, and is applied to chemiluminescence immunoassay system, and the luminescence system is
Direct chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, the reaction does not need the participation of enzyme, more cost-effective；
2nd, the acridinium ester chemiluminescent immunoassay system detection sensitivity that the present invention is selected is high, can reach 0.4 AU/mL, phase
5 times are at least improve than the sensitivity of other Anti-Scl-70 IgG detection methods；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention is selected is wide, can reach 3-190 AU/mL, other
Anti-Scl-70 IgG chemistry hair detection method the inspection range of linearity be 20-135 AU/mL；
4th, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention is selected is high, in batch and difference between batch is within 5%,
This is that other chemiluminescence immunoassay systems are unapproachable；
5th, chemiluminescence immunoassay system of the invention has realized quantifying for sample, soft to testing by built-in standard curve
Part, only needs test sample to directly obtain the concentration value of sample；
6th, chemiluminescence immunoassay system of the invention has realized full-automation, and the addition of reagent and sample has instrument complete entirely
Into operation is easier, reduces artificial error.
Brief description of the drawings
Fig. 1 is the Anti-Scl-70 IgG canonical plottings that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：Anti-Scl-70 IgG chemiluminescence immune detection reagent kit preparation methods
（1）It is prepared by the coated nanometer magnetic bead of Anti-Scl-70 IgG monoclonal antibody：
Take the magnetic particle of 50mg carboxylated（Particle diameter is 0.05-1um）Suspension, Magneto separate removes supernatant, and it is 5.5 to use 0.02 M, pH
MES buffer solutions are resuspended, add the EDC aqueous solution of 10 mg/mL of the new configurations of 0.5-2mL, activated magnetic beads surface carboxyl groups to add 3-5
Mg Anti-Scl-70 IgG monoclonal antibodies, suspension 2-10 h, Magneto separate, remove supernatant, with containing 2% BSA's at room temperature
0.1 M pH are that 8.0 Tris buffer solutions are resuspended to 1mg/mL, obtain the coated magnetic of Anti-Scl-70 IgG monoclonal antibody
Grain, every bottle of 5mL packing be stored in 4 DEG C it is standby.
（2）The preparation of the acridinium ester of mouse anti-human igg mark：
The mouse anti-human igg of 50 uL 25mg/mL is taken, the carbonate buffer solution of 150 uL 0.1-0.2 M pH 9.0-9.5 is added,
Mix, the acridinium ester for being subsequently adding the mg/mL of 1-2 uL 5 is mixed, at room temperature lucifuge reaction, is taken out after 1-2 h, with 2 mL's
Zeba is centrifuged desalting column desalting processing, is processed with pure water and TBS buffer solutions respectively first in desalination processes, is eventually adding
The acridine ester solution of the mouse anti-human igg mark for obtaining, liquid to the preservation collected in centrifuge tube is in control mouse anti-human igg mark
Acridinium ester, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）Anti-Scl-70 IgG calibrates the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）Anti-Scl-70 IgG is matched somebody with somebody
Concentration is set to for 0.1 AU/mL, 13.1 AU/mL, 26.0 AU/mL, 51.3 AU/mL, 102.2 AU/mL, 202.3 AU/mL,
Every bottle of 0.5 mL packing, 4 DEG C save backup.
(4) preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 80uL mass fractions are 20% is sequentially added2O2), 1.0 grams of sodium azide, 1.5
Gram polysorbas20, shakes up rear lucifuge storage.
(5) preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.6 gram of NaOH, 0.5 gram of PC300,0.5g sodium azide, 1.5 grams of Triton are sequentially added
405, shake up rear lucifuge storage.
Embodiment 2：Anti-Scl-70 IgG chemical luminous immune detection methods：
The present invention is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter, and method of the present invention pattern is indirect method, i.e.,
Instrument sequentially adds the sample of 5 uL（1:20 dilutions）, 50 uL the coated magnetic particle of Anti-Scl-70 IgG monoclonal antibody
After reacting 10 min, cleaning adds the Anti-Scl-70 IgG test dilutions of 100 uL and the mouse anti-human igg mark of 50 uL
Acridinium ester, reaction 10 min after, carry out Magneto separate, reactant mixture is sent into darkroom by instrument, sequentially add 50uL chemistry hair
Light preexciting liquid, 50uL chemiluminescences exciting liquid carry out luminescence-producing reaction, finally record luminous intensity, from standard curve calculate by
The Anti-Scl-70 IgG content of test sample product.
Embodiment 3：Anti-Scl-70 IgG chemiluminescence immune detection reagent kit performance evaluations
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, Anti-Scl-70 IgG chemiluminescence immunoassays chromatography reagent is calculated
The sensitivity of box, the sensitivity tried to achieve is 0.4AU/ mL.
Linear detection：
It is that 13.1 AU/mL, 26.0 AU/mL, 51.3 AU/mL, 102.2 AU/mL, 202.3 AU/mL standard items do to concentration
Linear analysis, calculates linearly dependent coefficient, r=0.9996, in addition, the kit resists the line of Scl-70 IgG antibody sample detections
Property scope be 3-190 AU/mL.
Precision is determined：
Concentration is taken for two Anti-Scl-70 IgG samples of 43.3 AU/mL and 107.7AU/mL, each concentration of each sample is each
Do 3 it is parallel, detected with three batches of kits, calculate in kit batch and difference between batch, as a result show in the kit batch and
Difference between batch is respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：It is combined with bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, sweet
Grease, adding proportion is according to 1:20 are carried out, and are determined pooled serum respectively and be with the addition of the survey of pooled serum after various chaff interferences
Value, calculates deviation therebetween, with ± 10% for tolerance interval.Result shows that interference reaches the file of NCCLS
Standard, can be used for the accurate evaluation of clinical labororatory's Anti-Scl-70 IgG situations.
Embodiment 4:The Sensitivity comparison experiment of Anti-Scl-70 IgG chemiluminescence immune detection reagent kits
It is respectively the calibration object or sample of 0AU/mL to concentration with chemical luminescence detection method and traditional enzyme linked immunosorbent assay
Dilution is detected that replication 20 times draws 20 RLU values of measurement result for sample（Relative light unit）, calculate it
Average value（M）And standard deviation（SD）, M+2SD is drawn, luminous value substitution calibration curve is calculated corresponding concentration value.Adopt
The concentration value obtained with chemical luminescence detection method is 0.4AU/mL, relative to traditional enzyme linked immunosorbent assay LDL
2.5AU/mL, improves about 6 times.
Claims (10)
1. a kind of Anti-Scl-70 IgG chemiluminescence immune detection reagent kits, the kit includes：Anti-Scl-70
IgG monoclonal antibody is coated-nanometer magnetic microsphere, chemiluminescent labels, Chemoluminescent substrate, Anti-Scl-70 IgG
Calibration product.
2. kit according to claim 1, it is characterised in that the Anti-Scl-70 IgG monoclonal antibody coating
Solid phase carrier be magnetic particle.
3. kit according to claim 1, it is characterised in that the Anti-Scl-70 IgG monoclonal antibody coating
Solid phase carrier for carboxylated particle diameter be 0.05-1um magnetic particles.
4. kit according to claim 1, it is characterised in that the chemiluminescent labels are acridinium ester, acridinium ester
Sulfonamide, acridinium ester toluenesulfonamide, acridinium ester are to methylsulfonamides, acridinium ester trimethyl fluoride sulfonyl amine.
5. kit according to claim 1, it is characterised in that the preferred acridinium ester of chemiluminescent labels.
6. kit according to claim 1, it is characterised in that the Chemoluminescent substrate is excited including chemiluminescence
Liquid, chemiluminescence preexciting liquid.
7. kit according to claim 1, it is characterised in that the chemiluminescence preexciting liquid is mass fraction
0.005% ~ 0.5% hydrogen peroxide (H2O2) solution, exciting liquid is the NaOH of 0.005mol/L ~ 0.025mol/L
(NaOH) solution.
8. kit according to claim 1, it is characterised in that the Anti-Scl-70 IgG calibrations product are to use standard
Savor buffer solution and Anti-Scl-70 IgG is configured to concentration for 0.1 AU/mL, 13.1 AU/mL, 26.0 AU/mL, 51.3 AU/
ML, 102.2 AU/mL, 202.3 AU/mL, 4 DEG C save backup.
9. kit according to claim 1, it is characterised in that the preparation method of the kit, it is characterised in that bag
Include preparation, the preparation of the acridinium ester of mouse anti-human igg mark, the change of the coated magnetic particle of Anti-Scl-70 IgG monoclonal antibody
Learn the preparation that product are calibrated in the preparation of luminous substrate liquid, Anti-Scl-70 IgG.
10. according to claim 1 and claim 9 kit preparation method, it is characterised in that comprise the following steps：
1）The preparation of the coated magnetic particle of Anti-Scl-70 IgG monoclonal antibody：
The nanometer magnetic bead suspension of carboxylated is taken, Magneto separate removes supernatant, and MES buffer solutions are resuspended, add the EDC aqueous solution, activate magnetic
Bead surface carboxyl, adds Anti-Scl-70 IgG monoclonal antibody, at room temperature suspension 2-10 h, and Magneto separate removes supernatant,
Tris buffer solutions are resuspended, obtain the coated magnetic particle of Anti-Scl-70 IgG monoclonal antibody；Optionally, carboxylated nano magnetic
A diameter of 0.1 μm ~ 2.0 μm of pearl；MES buffer concentrations are 10mM ~ 100mM, pH 5.5 ~ 8.5；
2）The preparation of the acridinium ester of mouse anti-human igg mark：
Mouse anti-human igg is taken, carbonate buffer solution is added, mixed, be subsequently adding acridinium ester mixing, at room temperature lucifuge reaction, 1-2 h
After take out, be centrifuged desalting column desalting processing, processed with pure water and TBS buffer solutions respectively first in desalination processes, finally
The acridine ester solution of the mouse anti-human igg mark that addition is obtained, liquid to the preservation collected in centrifuge tube is in control mouse anti-human igg mark
The acridinium ester of note；
3）Anti-Scl-70 IgG calibrates the preparation of product：
Anti-Scl-70 IgG is configured to concentration for 0.1 AU/mL, 13.1 AU/mL, 26.0 AU/ with standard items buffer solution
ML, 51.3 AU/mL, 102.2 AU/mL, 202.3 AU/mL, 4 DEG C save backup；
4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 0.5 ~ 100uL mass fractions are 20% is sequentially added2O2), 0.5 ~ 5 gram prevent
Rotten agent, 0.5 ~ 5 gram of surfactant, shake up rear lucifuge storage；Optionally, preservative be commercialization sodium azide, PC300,
Surfactant is polysorbas20, Tween 80, Triton X100, Triton 405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.2 ~ 1 gram of NaOH, 0.5 ~ 5 gram of preservative, 0.5 ~ 5 gram of surface is sequentially added and is lived
Property agent, shake up the storage of rear lucifuge；Optionally, preservative is commercialization sodium azide, PC300, and surfactant is polysorbas20, tells
Temperature 80, Triton X100, Triton 405.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201620680296 | 2016-06-30 | ||
| CN2016206802960 | 2016-06-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113461822A (en) * | 2021-05-21 | 2021-10-01 | 深圳市新产业生物医学工程股份有限公司 | Scl-70 antibody or binding fragment thereof, screening method therefor, and detection kit comprising same |
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| CN105277689A (en) * | 2015-11-17 | 2016-01-27 | 苏州浩欧博生物医药有限公司 | Reagent kit and method for detecting total serum IgE through nano magnetic particle chemiluminescence method |
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2016
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750492A (en) * | 2008-12-04 | 2010-06-23 | 上海裕隆生物科技有限公司 | Self-immunity hepatitis detection protein chip and kit thereof |
| CN103954779A (en) * | 2014-03-12 | 2014-07-30 | 长春迪瑞医疗科技股份有限公司 | Testosterone detection reagent based on microparticle chemiluminescence immunoassay technology |
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| CN113461822A (en) * | 2021-05-21 | 2021-10-01 | 深圳市新产业生物医学工程股份有限公司 | Scl-70 antibody or binding fragment thereof, screening method therefor, and detection kit comprising same |
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Application publication date: 20170531 |